Oligonucleotide clamps having diagnostic applications

ABSTRACT

Compounds referred to herein as oligonucleotide clamps are provided that stably bind to target polynucleotides in a sequence-specific manner. The oligonucleotide clamps comprise one or more oligonucleotide moieties capable of specifically binding to a target polynucleotide and one or more pairs of binding moieties covalently linked to the oligonucleotide moieties. In accordance with the invention, upon annealing of the oligonucleotide moieties to the target polynucleotide, the binding moieties of a pair are brought into juxtaposition so that they form a stable covalent or non-covalent linkage or complex. The interaction of the binding moieties of the one or more pairs effectively clamps the specifically annealed oligonucleotide moieties to the target polynucleotide.

The invention relates generally to oligonucleotides and their use asprobes and therapeutic agents, and more particularly, to modifiedoligonucleotides whose ends are capable of spontaneously forming astable ring structure whenever such oligonucleotide specifically bindsto a target polynucleotide.

BACKGROUND

The unpredictability and expense of conventional drug discovery has ledto the exploration of several drug discovery approaches that promisemore systematic and/or rapid identification candidate compounds fortesting in biological assays and disease models. Examples of suchapproaches include selection of small peptides from a synthetic orrecombinant peptide libraries, e.g. Pitrung et al, U.S. Pat. No.5,143,854; Geysen et al, J. Immunol. Meth., 102:259-274 (1987); Lam etal, Nature, 354:82-84 (1991); Scott et al, Science, 249:386-390 (1990);the construction and selection of human or humanized antibodies fromrecombinant antibody libraries, e.g. Riechmann et al, Nature,332:323-327 (1988); Winter and Milstein, Nature, 349:293-299 (1991 );selection of aptamers or ribozymes from random sequence polynucleotidelibraries, e.g. Ellington and Szostak, Nature, 346:818-822 (1990);Blackwell et al, Science, 250:1104-1110 (1990); Tuerk et al, Science,249:505-510 (1990); Joyce, Gene, 82:83-87 (1989); Cech et al, U.S. Pat.No. 4,987,071; Haseloffet al, Nature, 334:585-591 (1988); and the use ofantisense oligonucleotides, e.g. Uhlmann and Peyman, Chemical Reviews,90:543-584 (1990); Goodchild, Bioconjugate Chemistry, 1:165-187 (1990);Helene et al, Biochim. Biophys. Acta, 1049:99-125 (1990); Cohen, Ed.,Oligonucleotides: Antisense Inhibitors of Gene Expression (MacmillanPress, New York, 1989); Crooke, Ann. Rev. Pharmacol. Toxicol.,32:329-376 (1992); McManaway et al, Lancet, Vol. 335, pgs. 808-811(1990); Bayever et al, Antisense Research and Development, 2:109-110(1992); Manson et al, Lymphokine Research, Vol. 9, pgs. 35-42 (1990);Lisziewicz et al, Proc. Natl. Acad. Sci., 90:3860-3864 (1993); Miller,Biotechnology, Vol. 9, pgs. 358-362. (1991 ); Chiang et al, J. Biol.Chem., Vol. 266, pgs. 18162-18171 (1991); Calabretta, Cancer Research,Vol. 51, pgs. 4505-4510 (1991); and the like.

Of the cited examples, the antisense approach presents a compellingadvantage of not requiring one or more initial screening steps toidentify candidate compounds capable of binding to a predeterminedtarget. Specific binding is achieved by providing an oligonucleotide oran analog thereof capable of forming a stable duplex or triplex with atarget nucleotide sequence based on Watson-Crick or Hoogsteen binding,respectively. Thus, as soon as the sequence of a target polynucleotideis determined, the structure of candidate antisense compounds is alsodetermined. The specifically bound antisense compound then eitherrenders the respective targets more susceptible to enzymaticdegradation, blocks translation or processing, or otherwise blocks orinhibits the function of a target polynucleotide.

Another advantage of the antisense approach has been the development ofreliable and convenient methods for solid phase synthesis ofpolynucleotides and analogs thereof, e.g. Caruthers, Science, Vol. 230,pgs 281-285 (1985); Beaucage et al, Tetrahedron, 48: 2223-2311 (1992);and Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach(1RL Press, Oxford, 1991 ). In particular, the availability of syntheticoligonucleotides and a variety of nuclease-resistant analogs, e.g.phosphorothioates, methylphosphonates, and the like, has encouragedinvestigation of antisense compounds for treating a variety ofconditions associated with the inappropriate expression of indigenousand/or exogenous genes, such as described in the references cited above.

Notwithstanding the many hurdles that have been overcome in the courseof developing antisense compounds, several significant uncertaintiesstill stand in the way of their widespread adoption as drugs. One suchuncertainty concerns the degree of specificity of antisenseoligonucleotides under physiological conditions. Antisenseoligonucleotides could be non-specific in at least two Senses: (i)duplex or triplex formation may lack specificity, e.g. non-perfectlymatched duplexes may form--leading to the unwanted inhibition ofnon-target polynucleotides, and (ii) the moieties not directly involvedin base pairing, e.g. the backbone or other appendant groups, mayinteract non-specifically with other cellular components leading toundesired side effects, e.g. Woolf et al, Proc. Natl. Acad. Sci.,89:7305-7309 (1992); Matsukura et al, Proc. Natl. Acad. Sci.,4:7706-7710 (1987); and the like. In regard to first type ofnonspecificity, it has been observed that duplexes involving longeroligonucleotides tend to be more tolerant of mismatches--and hence, lessspecific--than duplexes involving shorter oligonucleotides, e.g. Younget al, Nucleic Acids Research, 19:2463-2470 (1991). In regard to thesecond type of nonspecificity, such activity may not be surprising inview of the large body of work on the use of polyanions, in particularhomopolymeric polynucleotides, as anti-viral compounds, e.g. Levy,Chapter 7, in Stringfellow, editor, Interferon and Interferon Inducers(Marcel Dekker, New York, 1980). Interestingly, increased activity--andwith some polyanions increased toxicity--was observed with increasedpolymer size.

The uncertainty over nonspecific binding has led to the exploration ofseveral ways to modify oligonucleotides to enhance duplex or triplexstability of antisense compounds. One approach has been to couple duplexor triplex intercalating moieties to the antisense oligonucleotide, e.g.Park et al, Proc. Natl. Acad. Sci., 89:6653-6657 (1992); Stein et al,Gene, 72:333-341 (1988); Mergny et al, Science, 256:1681-1684 (1992);Miller, International application PCT/US92/03999; and the like. Anotherapproach involves the use of circular oligonucleotides, which areexonuclease resistant and have been shown to melt from single-strandedtargets at substantially higher temperatures than linearoligonucleotides when binding involves both Watson-Crick and Hoogsteenbase pairing, e.g. Prakash and Kool, J. Am. Chem. Soc., 114:3523-3527(1992).

Additional approaches for enhancing specificity and binding strengthwould be highly useful for DNA-based therapeutics and diagnosticapplications of nucleic acids.

SUMMARY OF THE INVENTION

The invention relates to compounds capable of forming stable circularcomplexes and/or covalently closed macrocycles after specificallybinding to a target polynucleotide. Generally, compounds-of theinvention comprise one or more oligonucleotide moieties capable ofspecifically binding to a target polynucleotide and one or more pairs ofbinding moieties covalently linked to the oligonucleotide moieties. Inaccordance with the invention, upon annealing of the oligonucleotidemoieties to the target polynucleotide, the binding moieties of a pairare brought into juxtaposition so that they form a stable covalent ornon-covalent linkage or complex. The interaction of the binding moietiesof the one or more pairs effectively clamps the specifically annealedoligonucleotide moieties to the target polynucleotide.

In one aspect, compounds of the invention comprise a first bindingmoiety, a first oligonucleotide moiety, a hinge region, a secondoligonucleotide moiety, and a second binding moiety, for example, asrepresented by the particular embodiment of the following formula:

    X--OL1--G--OL2--Y

wherein OL1 and OL2 are the first and second oligonucleotide moieties, Gis the hinge region, X is the first binding moiety and Y is the secondbinding moiety such that X and Y form a stable covalent or non-covalentlinkage or complex whenever they are brought into juxtaposition by theannealing of the oligonucleotide moieties to a target polynucleotide, asillustrated diagrammatically in FIG. 1a. Preferably, in this embodiment,one of OL1 and OL2 forms a duplex through Watson-Crick type of bindingwith the target polynucleotide while the other of OL1 and OL2 forms atriplex through Hoogsteen or reverse Hoogsteen type of binding. WheneverX and Y form a covalent linkage, the compound of the invention forms amacrocycle of the following form: ##STR1## wherein "XY" is the covalentlinkage formed by the reaction of X and Y.

In another aspect, compounds of the invention comprise a first bindingmoiety, a first, second, and third oligonucleotide moiety, a first andsecond hinge region, and a second binding moiety, for example, asrepresented by the particular embodiment of the following formula:

    X--OL1--G.sub.1 --OL2--G.sub.2 --OL3

wherein X and Y are described as above, G₁ and G₂ are the first andsecond hinge regions, and OL1, OL2, and OL3 are the first through thirdoligonucleotide moieties. Preferably, the sequences of OL1, OL2, and OL3are selected so that OL1 and OL2 and OL3 form triplex structures withthe target polynucleotide, as diagrammatically illustrated in FIG. 1b.Whenever X and Y form a covalent linkage, the compound of the inventionforms a macrocycle of the following form: ##STR2## wherein "XY" is thecovalent linkage formed by the reaction of X and Y.

In yet another aspect, the oligonucleotide clamps of the invention arecompositions of two or more components, e.g. having the form:

    X--OL1--W and Y--OL2--Z

wherein X, Y, W, and Z are defined as X and Y above. In this embodiment,the hinge region is replaced by additional complex-forming moieties Wand Z. As above, one of OL1 and OL2 undergoes Watson-Crick type ofbinding while the other undergoes Hoogsteen or reverse Hoogsteen type ofbinding to a target polynucleotide, as shown diagrammatically in FIG.1c. Similarly, whenever X and Y and W and Z form covalent linkages,compounds X--OL1--W and Y--OL2--Z form a macrocycle of the followingform: ##STR3## depending on the selection of OL1 and OL2.

Preferably, compounds of the invention are capable of forming covalentlyclosed macrocycles or stable circular complexes topologically linked toa target polynucleotide.

The invention provides compounds capable of specifically binding topredetermined target polynucleotides with superior stability thancurrently available probes and antisense compounds. Compounds of theinvention are employed either as antisense or antiogene compounds toinhibit the function of a polynucleotide whose expression orparticipation in a regulatory function is associated with a diseasestate or as probes for detecting the presence of a targetpolynucleotide. The invention includes the oligonucleotide clamps per seas well as pharmaceutical compositions and kits for particularapplications.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a-c diagrammatically illustrate how three separate embodiments ofthe invention bind or "clamp" a target polynucleotide.

FIG. 2a-b diagrammatically illustrates the concept of "topologicallylinked."

FIG. 3 illustrates the results of the inhibition of the cytotoxiceffects of HIV in an ATH8 cell assay.

Definitions

"Topologically linked" in reference to compounds of the invention refersto the relative configuration of target polynucleotide andoligonucleotide clamp wherein the oligonucleotide clamp forms a closedcircular complex or macrocycle enclosing the target polynucleotidestrand, as shown diagrammatically in FIGS. 2a and 2b. In FIG. 2a,oligonucleotide clamp 20 is topologically linked to targetpolynucleotide 10. In FIG. 2b, oligonucleotide 20 is topologicallydisjoint from target polynucleotide 10.

"Stable" in reference to the formation of a covalent linkage and/ornon-covalent complex between binding moieties means that meltingtemperature of the oligonucleotide clamp incorporating the given pair(s)of binding moieties and its target polynucleotide is increased by atleast fifty percent over the melting temperature of oligonucleotidemoieties of the clamp alone, wherein melting temperature is measured bystandard techniques, e.g. half maximum of 260 nm absorbance v.temperature as described more fully below.

"Linkage" in reference to the reaction of binding moieties includes bothcovalent linkages and non-covalent complexes.

The term "oligonucleotide" as used herein includes linear oligomers ofnatural or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, α-anomeric forms thereof,polyamide nucleic acids, and the like, capable of specifically bindingto a target polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, Hoogsteen or reverse Hoogsteen types of base pairing, or thelike. Usually monomers are linked by phosphodiester bonds or analogsthereof to form oligonucleotides ranging in size from a few monomericunits, e.g. 3-4, to several hundreds of monomeric units. Whenever anoligonucleotide is represented by a sequence of letters, such as"ATGCCTG," it will be understood that the nucleotides are in 5'->3'order from left to right and that "A" denotes deoxyadenosine, "C"denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotesthymidine, unless otherwise noted. Analogs of phosphodiester linkagesinclude phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, and the like.

As used herein, "nucleoside" includes the natural nucleosides, including2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described generallyby Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogsinclude synthetic nucleosides designed to enhance binding properties,e.g. stability, specificity, or the like, such as disclosed by thereferences cited herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to oligonucleotide clamps that are capable ofbinding to a specific region of a target polynucleotide. The clampingaspect of the compounds is achieved by the formation of stable linkagesor complexes between binding moieties after they are brought intoproximity by specific binding of the one or more oligonucleotidemoieties to a target polynucleotide. Preferably, oligonucleotidemoieties of the compounds of the invention are selected so that theysimultaneously undergo Watson-Crick and Hoogsteen types of binding withspecific regions of a target polynucleotide.

In embodiments where triplex formation is desired, there are constraintson the selection of target sequences. Generally, third strandassociation via Hoogsteen type of binding is most stable alonghomopyrimidine-homopurine tracks in a double stranded target. Usually,base triplets form in T-A*T or C-G*C motifs (where "-" indicatesWatson-Crick pairing and "*" indicates Hoogsteen type of binding);however, other motifs are also possible. For example, Hoogsteen basepairing permits parallel and antiparallel orientations between the thirdstrand (the Hoogsteen strand) and the purine-rich strand of the duplexto which the third strand binds, depending on conditions and thecomposition of the strands. There is extensive guidance in theliterature for selecting appropriate sequences, orientation, conditions,nucleoside type (e.g. whether ribose or deoxyribose nucleosides areemployed), base modifications (e.g. methylated cytosine, and the like)in order to maximize, or otherwise regulate, triplex stability asdesired in particular embodiments, e.g. Roberts et al, Proc. Natl. Acad.Sci., 88:9397-9401 (1991); Roberts et al, Science, 258:1463-1466 (1992);Distefano et al, Proc. Natl. Acad. Sci., 90:1179-1183 (1993); Mergny etal, Biochemistry, 30:9791-9798 (1991); Cheng et al, J. Am. Chem. Soc.,114:4465-4474 (1992); Beal and Dervan, Nucleic Acids Research,20:2773-2776 (1992); Beal and Dervan, J. Am. Chem. Soc., 114:4976-4982(1992); Giovannangeli et al, Proc. Natl. Acad. Sci., 89: 8631-8635(1992); Moser and Dervan, Science, 238:645-650 (1987); McShan et al, J.Biol. Chem., 267:5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci.,89:3840-3844 (1992); Blume et al, Nucleic Acids Research, 20:1777-1784(1992); and the like. Generally, after one of the oligonucleotidemoieties forms a Watson-Crick duplex with a pyrimidine-rich orpurine-rich track in a target polynucleotide, the remainingoligonucleotide components bind to the major groove of the duplex toform a triplex structure.

Selection of particular oligonucleotide sequences for triplex formationcan also be carded out empirically, for example, through aptamerscreening, or like process, where candidate oligonucleotide moieties areselected on the basis of binding strength to an immobilized doublestranded target, e.g. Ellington and Szostak, Nature, 346:818-822 (1990);Toole et al, International application PCT/US92/01383; and the like.

Target polynucleotides may be single stranded or double stranded DNA orRNA; however, single stranded DNA or RNA target polynucleotides arepreferred.

Preferably, stability of oligonucleotide clamp/target polynucleotidecomplexes are determined by way of melting, or strand dissociation,curves. The temperature of fifty percent strand dissociation is taken asthe melting temperature, T_(m), which, in turn, provides a convenientmeasure of stability. T_(m) measurements are typically carried out in asaline solution at neutral pH with target and clamp concentrations atbetween about 1.0-2.0 μM. Typical conditions are as follows: 150 mM NaCland 10 mM MgCl₂ in a 10 mM sodium phosphate buffer (pH 7.0) or in a 10mM Tris-HCl buffer (pH 7.0); or like conditions. Data for melting curvesare accumulated by heating a sample of the oligonucleotide clamp/targetpolynucleotide complex from room temperature to about 85°-90° C. As thetemperature of the sample increases, absorbance of 260 nm light ismonitored at 1° C. intervals, e.g. using a Cary (Australia) model 1E ora Hewlett-Packard (Palo Alto, Calif.) model HP 8459 UV/VISspectrophotometer and model HP 89100A temperature controller, or likeinstruments.

The oligonucleotide moieties of the invention are synthesized byconventional means on a commercially available automated DNAsynthesizer, e.g. an Applied Biosystems (Foster City, Calif.) model380B, 392 or 394 DNA/RNA synthesizer. Preferably, phosphoramiditechemistry is employed, e.g. as disclosed in the following references:Beaucage and Iyer, Tetrahedron, 48:2223-2311 (1992); Molko et al, U.S.Pat. No. 4,980,460; Koster et al, U.S. Pat. No. 4,725,677; Caruthers etal, U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679; and the like.For therapeutic use, nuclease resistant backbones are preferred. Manytypes of modified oligonucleotides are available that confer nucleaseresistance, e.g. phosphorothioate, phosphorodithioate, phosphoramidate,or the like, described in many references, e.g. phosphorothioates: Stecet al, U.S. Pat. No. 5,151,510; Hirschbein, U.S. Pat. No. 5,166,387;Bergot, U.S. Pat. No. 5,183,885; phosphoramidates: Froehler et al,International application PCT/US90/03138; and for a review of additionalapplicable chemistries: Uhlmann and Peyman (cited above). In someembodiments it may be desirable to employ P-chiral linkages, in whichcase the chemistry disclosed by Stec et al, European patent application92301950.9, may be appropriate.

The length of the oligonucleotide moieties is sufficiently large toensure that specific binding will take place only at the desired targetpolynucleotide and not at other fortuitous sites. The upper range of thelength is determined by several factors, including the inconvenience andexpense of synthesizing and purifying oligomers greater than about 30-40nucleotides in length, the greater tolerance of longer oligonucleotidesfor mismatches than shorter oligonucleotides, and the like. Preferably,the oligonucleotide moieties have lengths in the range of about 6 to 40nucleotides. More preferably, the oligonucleotide moieties have lengthsin the range of about 12 to 25 nucleotides.

Hinge regions consist of nucleosidic or non-nucleosidic polymers whichpreferably facilitate the specific binding of the monomers of theoligonucleotide moieties with their complementary nucleotides of thetarget polynucleotide. Generally, the oligonucleotide moieties may beconnected to hinge regions and/or binding moieties in either 5'->3' or3'->5' orientations. For example, in the embodiment described abovecomprising a first binding moiety, a first oligonucleotide moiety, ahinge region, a second oligonucleotide moiety, and a second bindingmoiety, the oligonucleotide moieties may have any of the followingorientations:

    X--(5')N.sub.1 N.sub.2 N.sub.3 --. . . --N.sub.j (3')--G--(5')N.sub.1 N.sub.2 N.sub.3 --. . . --N.sub.k (3')--Y

OR

    X--(5')N.sub.1 N.sub.2 N.sub.3 --. . .--N.sub.j (3')--G--(3')--G--(3')N.sub.k N.sub.k--1 N.sub.k--2 --. . .--N.sub.1 (5')--Y

OR

    X--(3')N.sub.j N.sub.j--1 N.sub.j--2 --. . .--N.sub.1 (5')--G--(5')N.sub.1 N.sub.2 N.sub.3 --. . .--N.sub.k (3')--Y

OR

    X--(3')N.sub.j N.sub.j--1 N.sub.j--2 --. . .--N.sub.1 (5')--G--(3')N.sub.k N.sub.k--1 N.sub.k--2 --. . .--N.sub.1 (5')--Y

wherein N₁ N₂ N₃ --. . . --N_(k) and N₁ N₂ N₃ --. . .--N_(j) are k-merand j-mer oligonucleotide moieties in the indicated orientations.

Preferably, the hinge region has the general form:

    --(M--L).sub.n --

wherein M may be an inert non-sterically hindering spacer moiety servingto connect the oligonudeotide moieties, wherein the M's and L's in anygiven chain may be the same or different. Alternatively, one or more ofmonomers M may contain reactive functionalities for attaching labels;oligonucleotides or other binding polymers for hybridizing or binding toamplifier strands or structures, e.g. as described by Urdea et al, U.S.Pat. No. 5,124,246 or Wang et al, U.S. Pat. No. 4,925,785; "hooks", e.g.as described in Whiteley et al, U.S. Pat. No. 4,883,750; or other groupsfor affecting solubility, cellular delivery, promotion of duplex and/ortriplex formation, such as intercalators, alkylating agents, and thelike. Preferably, the hinge regions provide a spacer of about 16-28angstroms between the termini of the oligonucleotides.

Preferably, L is a phosphorus(V) linking group which may bephosphodiester, phosphotfiester, methyl or ethyl phosphonate,phosphorothioate, phosphorodithioate, phosphoramidate, or the like.Generally, linkages derived from phosphoramidite precursors arepreferred so that compounds of the invention can be convenientlysynthesized with commercial automated DNA synthesizers, e.g. AppliedBiosystems, Inc. (Foster City, Calif.) model 394, or the like.

n may vary significantly depending on the nature of M and L. Generally,n will vary from 1 for M comprising alkyl, alkenyl, and/or etherscontaining 10 or more carbon atoms, e.g. Salunkhe et al, J. Am. Chem.Sot., 114:8768-8772 (1992), to about 10 for M comprising alkyl, alkenyl,and/or ethers containing 2-3 carbon atoms. Preferably, for a hingemoiety consisting entirely of an alkyl chain (and linkage moieties),such alkyl chain contains form 8 to 15 carbon atoms, and morepreferably, from 9 to 12 carbon atoms. Preferably, for nucleoside-sizedmonomers, n varies between about 3 and about 10; and more preferably, nvaries between about 4 and 8.

Preferably, hinge moieties are synthesized using conventionalphosphoramidite and/or hydrogen phosphonate chemistries. The followingreferences disclose several phosphoramidite and/or hydrogen phosphonatemonomers suitable for use in the present invention and provide guidancefor their synthesis and inclusion into oligonucleotides: Newton et al,Nucleic Acids Research, 21:1155-1162 (1993); Griffin et al, J. Am. Chem.Sot., 114:7976-7982 (1992); Jaschke et al, Tetrahedron Letters,34:301-304 (1992); Ma et at, International application PCT/CA92/00423;Zon et al, International application PCT/US90/06630; Durand et at,Nucleic Acids Research, 18:6353-6359 (1990); Salunkhe et al, J. Am.Chem. Soc., 114:8768-8772 (1992); and the like.

In a preferred embodiment, M is a straight chain, cyclic, or branchedorganic molecular structure containing from 1 to 20 carbon atoms andfrom 0 to 10 heteroatoms selected from the group consisting of oxygen,nitrogen, and sulfur. More preferably, M is alkyl, alkoxy, alkenyl, oraryl containing from 1 to 16 carbon atoms; heterocyclic having from 3 to8 carbon atoms and from 1 to 3 heteroatoms selected from the groupconsisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl.Most preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1to 8 carbon atoms; glycosyl; or nucleosidyl.

A variety of binding moieties are suitable for use with the invention.Generally, they are employed in pairs, which for convenience here willbe referred to as X and Y. X and Y may be the same or different.Whenever the interaction of X and Y is based on the formation of stablehydrophobic complex, X and Y are lipophilic groups, including alkylgroups, fatty acids, fatty alcohols, steroids, waxes, fat-solublevitamins, and the like. Further exemplary lipophilic binding moietiesinclude glycerides, glyceryl ethers, phospholipids, sphingolipids,terpenes, and the like. In such embodiments, X and Y are preferablyselected from the group of steroids consisting of a derivatizedperhydrocyclopentanophenanthrene nucleus having from 19 to 30 carbonatoms, and 0 to 6 oxygen atoms; alkyl having from 6 to 16 carbon atoms;vitamin E; and glyceride having 20 to 40 carbon atoms. Preferably, aperhydrocyclopentanophenanthrene-based moiety is attached through thehydroxyl group, either as an ether or an ester, at its C3 position. Itis understood that X and Y may include a linkage group connecting it toan oligonucleotide moiety. For example, glyceride includesphosphoglyceride, e.g. as described by MacKellar et al, Nucleic AcidsResearch, 20:3411-3417 (1992), and so on. It is especially preferredthat lipophilic moieties, such as perhydrocyclopentanophenanthrenederivatives, be linked to the 5' carbon and/or the 3' carbon of anoligonucleotide moiety by a short but flexible linker that permits thelipophilic moiety to interact with the bases of the oligonucleotideclamp/target polynucleotide complex or a lipophilic moiety on the sameor another oligonucleotide moiety. Such linkers include phosphate (i.e.phosphodiester), phosphoramidate, hydroxyurethane, carboxyaminoalkyl andcarboxyaminoalkylphosphate linkers, or the like. Preferably, suchlinkers have no more than from 2 to 8 carbon atoms.

Binding moieties can be attached to the oligonucleotide moiety by anumber of available chemistries. Generally, it is preferred that theoligonucleotide be initially derivatized at its 3' and/or 5' terminuswith a reactive functionality, such as an amino, phosphate,thiophosphate, or thiol group. After derivatization, a hydrophilic orhydrophobic moiety is coupled to the oligonucleotide via the reactivefunctionality. Exemplary means for attaching 3' or 5' reactivefunctionalities to oligonucleotides are disclosed in Fung et al, U.S.Pat. No. 5,212,304; Connolly, Nucleic Acids Research, 13: 4485-4502(1985); Tino, International application PCT/US91/09657; Nelson et al,Nucleic Acids Research, 17:7187-7194 (1989); Stabinsky, U.S. Pat. No.4,739,044; Gupta et al, Nucleic Acids Research, 19:3019 (1991 ); Reed etal, International application PCT/US91/06143; Zuckerman et al, NucleicAcids Research, 15:5305 (1987); Eckstein, editor, Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991 ); Clontech1992/1993 Catalog (Clontech Laboratories, Palo Alto, CA); and likereferences.

Preferably, whenever X and Y form a covalent linkage, X and Y pairs mustreact specifically with each other when brought into juxtaposition, butotherwise they must be substantially unreactive with chemical groupspresent in a cellular environment. In this aspect of the invention, Xand Y pairs are preferably selected from the following group: when oneof X or Y is phosphorothioate, the other is haloacetyl, haloacyl,haloalkyl, or alkylazide; when one of X or Y is thiol, the other isalkyl iodide, haloacyl, or haloacetyl; when one of Y or Y is phenylazidethe other is phenylazide. More preferably, when one of X or Y isphosphorothioate, the other is haloacetyl, haloacyl, or haloalkyl,wherein said alkyl, acetyl, or acyl moiety contains from one to eightcarbon atoms.

Most preferably, when one of X or Y is phosphorothioate, the other ishaloacetyl. Most preferably, whenever one of X or Y is phosphorothioate,the other is bromoacetyl. These binding moieties form a covalentthiophosphoylacetylamino bridge, as shown below, selectively andefficiently at low concentrations, e.g. less than one μM, when reactedin an aqueous environment in the presence of a target polynucleotide:##STR4## wherein X is halo and N₁, N₂, N_(j) and N_(k) are nucleotidesof a j-mer and k-mer, respectively. Compound 1 can be prepared byN-succinimidyl haloacetate in N,N-dimethylformamide (DMF) with a3'-aminodeoxyribonucleotide precursor in a sodium borate buffer at roomtemperature. After about 35 minutes the mixture is diluted (e.g. with H₂O), desalted and, purified, e.g. by reverse phase HPLC. The3'-aminodeoxyribonucleotide precursor can be prepared as described inGryaznov and Letsinger, Nucleic Acids Research, 20:3403-3409 (1992) orTetrahedron Letters, 34: 1261-1264 (1993). Briefly, after deprotection,the 5' hydroxyl of a deoxythymidine linked to a support via a standardsuccinyl linkage is phosphitylated by reaction withchloro-(diisopropylethylamino)-methoxyphosphine in an appropriatesolvent, such as dichloromethane/diisopropylethylamine. After activationwith tetrazole, the 5'-phosphitylated thymidine is reacted with a5'-trityl-O-3'-amino-3'-deoxynucleoside to form a nucleoside-thymidinedimer wherein the nucleoside moieties are covalently joined by aphosphoramidate linkage. The remainder of the oligonucleotide issynthesized by standard phosphoramidite chemistry. After cleaving thesuccinyl linkage, the oligonucleotide with a 3'-terminal amino group isgenerated by cleaving the phosphoramidate link by acid treatment, e.g.80% aqueous acetic acid for 18-20 hours at room temperature.5'-trityl-O-3'-amino-3'-deoxynucleosides may be synthesized inaccordance with Glinski et al, J. Chem. Soe. Chem. Comm., 915-916(1970); Miller et al, J. Org. Chem. 29:1772 (1964); Zielinki and Orgel,Nucleic Acids Research, 13:2469-2484 (1985) and 15:169%1715 (1987);Ozols et al, Synthesis, 7:557-559 (1980); and Azhayev et al, NucleicAcids Research, 6: 625-643 (1979); which references are incorporated byreference.

5' monophosphorothioate 2 is formed as follows: A 5' monophosphate isattached to the 5' end of an oligonucleotide either chemically orenzymatically with a kinase, e.g. Sambrook et al, Molecular Cloning: ALaboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory, New York,1989). Preferably, as a final step in oligonucleotide synthesis, amonophosphate is added by chemical phosphorylation as described by Hornand Urdea, Tetrahedron Lett., 27:4705 (1986) (e.g. using commerciallyavailable reagents such as 5'Phosphate-ON^(TM) from ClontechLaboratories (Palo Alto, California)). The 5'-monophosphate is thensulfurized using conventional sulfurizing agents, e.g. treatment with a5% solution of S₈ in pyridine/CS₂ (1:1, v/v, 45 minutes at roomtemperature); or treatment with sulfurizing agent described in U.S. Pat.Nos. 5,003,097; 5,151,510; or 5,166,387. Preferably, the haloacetylaminoderivatized oligonucleotides are synthesized separately from unprotectedmonophosphorothioate groups.

Compounds of the invention can be employed as diagnostic probes todetect the presence of one or more target polynucleotides in a widerange of samples, including environmental samples, e.g. from publicwater supplies, samples from foodstuffs, and from other biologicalsamples, such as blood, saliva, semen, amniotic fluid, tissuehomogenates of plants or animals, or of human patients, and the like.The use of nucleic acid probes in human diagnostics, forensics, andgenetic analysis has been extensively reviewed. For example, thefollowing references describe many diagnostic applications of nucleicacid probes for which the present invention can be usefully employed:Caskey, Science 236:1223-1228 (1987); Landegren et al, Science,242:229-237 (1988); and Arnheim et al, Ann. Rev. Biochem., 61:131-156(1992). Moreover, there is extensive guidance in the literatureconcerning the selection of hybridization conditions, labeling means,and the like, which is applicable to the practice of the presentinvention, e.g. Wallace et al, Nucleic Acids Research 6:3543-3557(1979); Crothers et al, J. Mol. Biol. 9:1-9 (1964); Gotoh, Adv. Biophys.16:1-52 (1983); Wetruer, Critical Reviews in Biochemistry and MolecularBiology 26:227-259 (1991 ); Breslauer et al, Proc. Natl. Acad. Sci.83:3746-3750 (1986); Wolfet al, Nucleic Acids Research, 15:2911-2926(1987); McGraw et al, Biotechniques, 8:674-678 (1990), and the like.

Oligonucleotide clamps of the invention may be used in essentially anyof the known solution or solid phase hybridization formats, such asthose in which the analyte is bound directly to a solid phase, orsandwich hybridizations in which the analyte is bound to anoligonucleotide that is, in turn, bound to a solid phase.Oligonucleotide clamps having an oligonucleotide "tail" attached to ahinge region are particularly useful in conjunction with branchedpolymer amplification schemes, such as those disclosed by Urdea et al,U.S. Pat. No. 5,124,246; Wang et al, U.S. Pat. No. 4,925,785; and thelike. Urdea et al and Wang et al are incorporated by reference for theirdescription of such hybridization assays. In such embodiments, theoligonucleotide clamp serves as a highly stable "capture" probe bybinding to a target polynucleotide analyte of interest. Theoligonucleotide tail then hybridizes with a directly or indirectlylabeled amplifier strand or complex. Such tails are long enough to forma stable duplex with the amplifier strand. Preferably, such tails arebetween 18 and 60 nucleotides in length. Tails may also comprise asecond oligonucleotide clamp. That is, a dimer of oligonucleotide clampshaving different binding specificities can be used to tightly couple anamplifier complex to a target polynucleotide.

Preferably, oligonucleotide tails are coupled to hinge regions at anamino group which has been derivatized with bromoacetyl. Anoligonucleotide tail having either a 5' or 3' phosphorothioate group isthen reacted with the bromoacetyl group to form athiophosphorylacetylamino bridge, as described more fully above and inthe Examples below. Phosphoramidate linkages are introduced inaccordance with published procedures, e.g. Letsinger, U.S. Pat. No.4,958,013; Agrawal et al, Nucleic Acids Research, 18:5419-5423 (1990);or the like. By a similar procedure, dimers of oligonucleotide clampscan also be constructed.

Whenever oligonucleotide clamps of the invention are employed indiagnostic assays, or in other processes not requiring direct contactwith a patient, a wider range of binding moieties may be employed thanwould be possible for therapeutic use. In diagnostic and othernon-therapeutic applications, reaction of the binding moieties mayinvolve an activation step wherein one or both of the binding moietiesare activated or rendered reactive towards one another by exposure to anactivating agent or condensing agent, such as radiation, a reducingagent, an oxidizing agent, or the like. Exemplary, binding moietiesemploying activating agents include thiophosphoryl groups in thepresence of K₃ Fe(CN)₆ or KI₃, e.g. Gryaznov and Letsinger, NucleicAcids Research, 21: 1403-1408 (1993 ); phosphoryl and hydroxyl in thepresence of N-cyanoimidazole, e.g. Luebke et al, J. Am. Chem. Soc.,113:7447-7448 (1991); phosphoryl or amino group and hydroxyl in thepresence of cyanogen bromide, e.g. Sokolova et al, FEBS Letters,232:153-155 (1988); phosphoryl and hydroxyl groups in the presence ofspermine-5-(N-ethylimidazole)carboxamide and cyanoimidazole, e.g. Zuberet al, J. Am. Chem. Soc., 115: 4939-4940 (1993); and the like.

Kits incorporating oligonucleotide clamps can take a variety of formsdepending on the particular embodiment, the type of assay formatemployed, and the labeling scheme employed. Generally, kits of theinvention comprise an oligonucleotide clamp specific for a given targetpolynucleotide, a hybridization buffer, and a signal generation moiety.Kits of the invention may further comprise wash buffers for removingunbound label and/or oligonucleotide clamps, solid phase supports suchas derivatized magnetic beads, or the like; and prehybridization bufferscontaining blocking agents, e.g. Denhardt's solution, sonicated salmonsperm DNA, detergents such as 1% SDS, or the like, for minimizingnonspecific binding of oligonucleotide clamps or other nucleosidicbinding components, such as amplifier strands. An exemplaryhybridization buffer comprises the following reagents: 100-150 mM NaCI,10 mM MgCl₂, and 10 mM Tris-HCl (pH 7.0).

Signal generation moieties are molecular structures that directly orindirectly generate a signal, e.g. fluorescent, colorimetric,radioactive, or the like, that can be detected by conventional means.Direct signal generation means that the moiety producing a signal iscovalently linked to the oligonucleotide clamp, e.g. as with thecovalent attachment of a fluorescent dye, enzyme, or the like. Indirectsignal generation means that a particular moiety, e.g. anoligonucleotide tail on a hinge region, is one component of amulticomponent system that produces a signal. Preferably, the signalgeneration moiety comprises an oligonucleotide tail of about 12 to about50 nucleotides in length covalently attached to a hinge region of anoligonucleotide clamp. In one aspect of this preferred embodiment, asignal is generated indirectly by providing a second oligonucleotidewhich is complementary to the tail and which has a fluorescent dyecovalently attached. Attaching fluorescent dyes to oligonucleotides iswell known in the art, e.g. U.S. Pat. Nos. 4,997,828; 5,151,507;4,855,225; 5,188,934; Eckstein, editor (cited above); and the like.

Compounds of the invention can be employed as components ofpharmaceutical compositions. A variety of diseases and disorders can betreated by administration of a composition comprising compounds of theinvention. Viral diseases that can be treated by antisense inhibition ofnucleic acid expression include, but are not limited to, those caused byhepatitis B virus, cytomegalovirus, herpes simplex virus I or II, humanimmunodeficiency virus type I or II, influenza virus, respiratorysyncytial virus, and human papilloma virus. Malignancies which can betreated by administration of antisense compounds of the inventioninclude those known to be directly or indirectly caused by theinappropriate expression of one or more gene, such as cancers caused bythe inappropriate expression of oncogenes, e.g. myb, bcr-abl, kit, myc,ras, raf, abl, or the like. In such diseases, the compounds of theinvention are specifically targeted to aberrantly expressed genesassociated with the diseases, or to regulatory polynucleotides thatinteract with aberrantly transcribed or expressed genes, e.g. Aaronson,Science, Vol. 254, pgs. 1146-1153 (1991 ). Acute inflammatory and immunereactions, such as septic shock, eosinophilia, and the like, can also betreated with compounds of the invention, wherein inappropriately and/oraberrantly expressed cytokine genes are inhibited, e.g. Tracey et al,Nature, Vol. 330, pgs. 662-664 (1987), U.S. Pat. No. 5,055,447, andWaage et al, J. Exp. Meal., Vol. 169, pgs. 333-338 (1989)(antisenseTNF-α and/or TNF-I3); Starnes et al, J. Immunol., Vol. 145, pgs.4185-4191 (1990), and Fong et al, J. Immunol., Vol. 142, pgs. 2321-2324(amisense IL-6); Coffman et al, Science, Vol. 245 pgs. 308-310(antisense IL-5); Finkelman et al, J. Immunol., Vol. 141, pgs. 2335-2341(1988)(antisense IL-4); Young et al, Blood, Vol. 68, pgs. 1178-1181(1986)(antisense GM-CSF); and the like.

The components included in pharmaceutical compositions of the inventiondepend on several factors, including the nature of the disease orcondition being treated, the location of disease lesions, the mode ofdrug delivery and/or administration contemplated, the latter of whichcan include in vivo-administration by way of regional or systemicperfusion, topical application, intranasal administration,administration by implanted or transdermal sustained release systems,and the like, as well as ex vivo administration for use in bone marrowpurging. A preferred method of administration of oligonucleotide clampscomprises either regional or systemic perfusion. According to a methodof regional perfusion, the afferent and efferent vessels supplying theextremity containing a lesion, e.g. a cancerous lesion, are isolated andconnected to a low-flow perfusion pump in continuity with an oxygenatorand heat exchanger. The iliac vessels may be used for perfusion of thelower extremities. The axillary vessels are cannulated high in theaxilla for upper extremity lesions. A pharmaceutical compositioncontaining an oligonucleotide clamp is added to the perfusion circuit,and the perfusion is continued for an appropriate time period, e.g. anhour. Perfusion rates of from 100 to 150 ml/minute may be employed forlower extremity lesions, while half that rate should be employed forupper extremity lesions. Systemic heparinization may be used throughoutthe perfusion, and reversed after the perfusion is complete. Thisisolation perfusion technique permits administration of higher dosed ofchemotherapeutic agent than would otherwise be tolerated upon infusioninto the arterial or venous systemic circulation.

For systemic infusion, the oligonucleotide clamps are preferablydelivered via a central venous catheter, which is connected to anappropriate continuous infusion device. Indwelling catheters providelong term access to the intravenous circulation for frequentadministrations of drugs over extended periods of time.

Generally a pharmaceutical composition of the invention facilitates thedelivery of an effective amount of the active drug to a desired site ina manner consistent with patient safety and comfort. An effective amountof an oligonucleotide clamp depends on several factors, including thedisease or condition being treated, the method of administration, thescheduling of the administration, the condition of the patient, and thelike. Typically, a parentially administered dose will be in the range ofabout 1 μg/kg/day to about 100 mg/kg/day of patient body weight. A keyfactor in selecting an appropriate dose for a given condition or diseaseis the therapeutic result, as measure by standard criteria well known tothe medical practitioner, e.g. for ontological applications see: Cancer:Principles and Practice of Oneology, 3rd Edition, edited by V. T. DeVitaet al (Lippincott Company, Philadelphia, 1989).

Pharmaceutical compositions of the invention include a pharmaceuticalcarder that may contain a variety of components that provide a varietyof functions, including regulation of drug concentration, regulation ofsolubility, chemical stabilization, regulation of viscosity, absorptionenhancement, regulation of pH, and the like. For example, in watersoluble formulations the pharmaceutical composition preferably includesa buffer such as a phosphate buffer, or other organic acid salt,preferably at a pH of between about 7 and 8. For formulations containingweakly soluble oligonucleotide clamps, microemulsions may be employed,for example by using a nonionic surfactant such as Tween 80 in an amountof 0.04-0.05% (W/v), to increase solubility. Other components mayinclude antioxidants, such as ascorbic acid, hydrophilic polymers, suchas, monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, dextrins, chelating agents, such as EDTA,and like components well known to those in the pharmaceutical Sciences,e.g. Remington's Pharmaceutical Science, latest edition (Mack PublishingCompany, Easton, Pa.).

Compounds of the invention include the pharmaceutically acceptable saltsthereof, including those of alkaline earths, e.g. sodium or magnesium,ammonium or NX₄ ⁺, wherein X is C₁₋₄ alkyl. Other pharmaceuticallyacceptable salts include organic carboxylic acids such as acetic,lactic, tartaric, malic, isethionic, lactobionic, and succinic acids;organic sulfonic acids such as methanesulfonic, ethanesulfonic, andbenzenesulfonic; and inorganic acids such as hydrochloric, sulfuric,phosphoric, and sulfamic acids. Pharmaceutically acceptable salts of acompound having a hydroxyl group include the anion of such compound incombination with a suitable cation such as Na⁺, NH₄ ⁺, or the like.

Sustained release systems suitable for use with the pharmaceuticalcompositions of the invention include semi-permeable polymer matrices inthe form of films, microcapsules, or the like, comprising polylactides,copolymers of L-glutamic acid and gamma-ethyl-L-glutamate,poly(2-hydroxyethyl methacrylate), and like materials. Sustained releasesystems also include liposomally entrapped oligonucleotide clamps, e.g.as described in Liposome Technology, Vol. II, Incorporation of Drugs,Proteins, an Genetic Material (CRC Press).

EXAMPLE 1 Synthesis of Oligonucleotide Clamp having 3' and 5'Cholesterol

Binding Moieties for pol and nef genes of HIV

A series of oligonucleotide clamps were synthesized that havecholesterol moieties attached to either a 5' end, a 3' end, or to both a3' end and a 5' end. The 3' cholesterol was attached by firstconstructing a cholesterol-derivatized solid phase support followed byroutine oligonucleotide chain extension via phosphoramidite monomers ona conventional automated DNA synthesizer (Applied Biosystems model 394).The 5' cholesterol was attached in the final coupling step of thesynthesis by reacting cholesterol chloroformate with the terminalnucleotide having a 5' amino group or by coupling a cholesterolphosphoramidite with a terminal hydroxyl group, the former methodusually giving higher yields.

(1) A polymer supported oligonucleotide, 1 μmole scale, with terminal5'-amino group was treated with 2 ml of a 10% solution of cholesterylformate in chloroform/diisopropylethylamine (9:1, v:v) for 20 minutes atroom temperature. The polymer support was then washed with chloroformand acetonitrile, cleaved and aleprotected with concentrated ammonium (5hours at 55° C.), and purified by reverse phase HPLC.

(2) A polymer supported oligonucleotide, 1 μmole scale, with terminalhydroxyl group was treated with 250 μl of 0.1M solution of cholesterolphosphoramidite in chloroform and 250 μl of 0.45M solution of tetrazolein acetonitrile for 10-15 minutes at room temperature. The polymersupport was then washed with acetonitrile, cleaved and deprotected withconcentrated ammonium (5 hours at 55° C.), and purified by reverse phaseHPLC.

EXAMPLE 2 ATH8 Assay of Oligonucleotide Clamps Specific for pol and nefgenes of HIV

The oligonucleotides and oligonucleotide clamps listed in the Table Ibelow were synthesized in accordance with Example 1 for specific bindingto the following single stranded or double stranded targetpolynucleotides:

                                      TABLE I                                     __________________________________________________________________________    5'---AAAAGAAAAGGGGGGA---3'                                                    3'---TTTTCTTTTCCCCCCT---5'                                                    Double stranded DNA                                                           5'---AAAAGAAAAGGGGGGA---3'                                                    Single stranded RNA or DNA                                                    Designation                                                                         Sequence of Oligonucleotide Clamp*                                      __________________________________________________________________________    050A  5'-CACTTTTCM.sup.Me TTTTCCCCCCTCACACTCCCCCCTTTTCTTTTAC-Chol             050B  5'-Chol-CACTTTTC.sup.Me TTTTCCCCCCTCACACTCCCCCCTTTTCTTTTAC-Chol         051   5'-Chol-CACTTTTC.sup.Me TTTTC.sup.Me C.sup.Me C.sup.Me-                       C.sup.Me C.sup.Me C.sup.Me TCACACTCCCCCCTMCTTTTAC-Chol                  052A  5'-CACTTTTC.sup.Me TTTTCCCCCCTCACACT-                                         C.sup.Me C.sup.Me C.sup.Me C.sup.Me C.sup.Me C.sup.Me TTTTCTTTTAC-Ch          ol                                                                      052B  5'-Chol-CACTTTTC.sup.Me TTTTCCCCCCTCA-                                        CACTC.sup.Me C.sup.Me C.sup.Me C.sup.Me C.sup.Me C.sup.Me TTTTCTTTTA          C-Chol                                                                  053   5'-Chol-CACTTTTC.sup.Me TTTTGGGGGGTCACACTCCCCCCTTTTCTTTTAC-Chol         DL015 5'-CACTTTTCTTTTGGGGGGTCACACTCCCCCCTTTTCTTTTAC                           DL014 5'-CACTTTTCTTTTGGGGGGTCACACTCCCCCCTTTTCTTTTAC-Chol                      DL013 5'-Chol-CACTTTTCTTTTGGGGGGTCACACTCCCCCCTTTTCTTTTAC-Chol                 DL021 5'-Chol-TTTTCTTTTCACACTTTTCTTTTGGGGGGTCACACTCCCCCC-Chol                 DL022 5'-Chol-CACTTTTCTTTTCCCCCCTCACACACTCCCCCCTTTTCTTTTAC-Chol               DL023 5'-Chol-TTTTCTTTTCACACTTTTCTTTTCCCCCCTCACTCCCCCC-Chol                   __________________________________________________________________________     *"Chol" represents cholesterol and C.sup.Me represents 5methylated            cytidine.                                                                

The melting temperature of the following compounds were determined bycomputing the half maximum of the 260 nm absorption v. temperaturecurve, as discribed above: DL015: 39.0° C.; DL014: 58° C.; DL013: 68.0°C.; DL021: 67.5° C.; DL022: 67.5° C.; and control (two unconnectedoligonucleotides without binding moieties having sequences:

3'-TTTTCTTTTCCCCCCT-5' and 5' -TTTTC^(Me) TTTT(C^(Me))₆ -3'): 32.0° C.Each of the compounds of Table I were tested in an ATH8 cell .assay toassess the inhibition of the cytopathic effects of HIV infection on theATH8 cells. The ATH8 assay is described in the following reference:Matsukura, pages 285-304 in Antisense RNA and DNA (Wiley-Liss, N.Y.,1992).

The cytopathic effect examined in the assay was the lytic effect of HIVand/or drug on the ATH8 cells. A measure of the degree of viral activityis the degree to which cells are lysed at the end of a given time periodin culture. Protection against lysis is measured by the size of pelletformed after cells are harvested from culture and centrifuged, asdescribed in the references cited above. The results of the assays aresummarized in Table II and FIGS. 3a and 3b.

                  TABLE II                                                        ______________________________________                                                Inhibition of Inhibition of                                           Oligo-  Cytotoxic*    Cytotoxic*                                              nucleotide                                                                            Effects at    Effects at                                              Clamp   2 μM Concentration                                                                       8 μM Concentration                                                                       Toxicity*                                 ______________________________________                                        015     0             0             0                                         014     +++           +++           0                                         013     ++            ++            0                                         021     ++            ++            0                                         022     ++            --            +                                         025     ++            --            +                                         050A    ++            ++            0                                         050B    ++            --            +                                         051     +++           --            +                                         052A    ++            +++           0                                         052B    not tested    --            +                                         053     +             +             0                                         ______________________________________                                         *"0" indicates no inhibitory effect observed; number of +'s indicates         relative inhibitory effect; "--" indicates toxicity at that dose.             **"0" indicates no toxicity observed; "+" indicates some toxicity             observed.                                                                

FIGS. 3 shows the change in pellet size versus time under differentexperimental conditions for oligonucleotide clamp 014. Curves 1 and 2illustrate results from positive and negative controls, i.e. curve 1illustrates the results for uninfected ATH8 cells without drug (negativecontrol) and curve 2 illustrates the results for infected ATH8 cellswithout drug (positive control). Curves 3 and 4 illustrate results fromcultures containing 8 μM of igonucleotide clamp in infected anduninfected ATH8 cells, respectively. Curves 5 and 6 illustrate resultsfrom cultures containing 2 μM of oligonucleotide clamp in infected anduninfected ATH8 cells, respectively. FIG. 3 shows that clamp 014 has aclear and positive effect at both concentrations without toxicity.

EXAMPLE 3 Synthesis of Two-component Oligonucleotide Clamps withCholesterol Binding Moieties

The following two-component oligonucleotide clamps were synthesized bythe procedures described above and tested in the ATH8 assay describedabove: ##STR5##

EXAMPLE 4 Synthesis of Oligonucleotide Clamps with PhosphorothioateLinkages

The following oligonucleotide clamps containing phosphorothioatelinkages were synthesized by the procedures described above and testedin the ATH8 assay described above: ##STR6## wherein "ps" indicates thepresence of a phosphorothioate linkage.

EXAMPLE 5 Synthesis of Oligonucleotide Clamp with Cholesterol BindingMoieties and a polyethylene Glycol Hinge Region

The following oligonucleotide clamp having a non-nucleosidic hingeregion is synthesized as described above and employing the protectedethyl glycol phosphoramidites disclosed by Durand et al, Nucleic AcidsResearch, 18:6353-6359 (1990); and Rumney et al (cited above): ##STR7##wherein "p" indicates the presence of a phosphodiester linkage.

EXAMPLE 6 Synthesis of Oligonucleotide Clamp havingBromoacetyl/Phosphorothioate Binding Moieties

The following oligonucleotide clamp is synthesized by the proceduresdescribed above and tested in the ATH8 assay: ##STR8##

5'-bromoacetylamino oligonudeotides are prepared as follows: 15 μL of0.4M N-succinimidyl bromoacetate (e.g. Calbiochem) inN,N-dimethylformamide (DMF) is added to 4.9 A₂₆₀ units of a5'-amino-oligonucleotide precursor in 10 mL of 0.2M sodium borate bufferat room temperature. After about 35 minutes the mixture is diluted (0.5mL H₂ O), desalted by gel filtration on a NAP-5 column (Pharmacia),purified by reverse phase HPLC (as described below), and again desaltedto yield 4 A₂₆₀ units of 5'-bromoacetylamino oligonucleotide (elutiontime for reverse phase HPLC, 17.4 minutes; ion exchange HPLC, 17.4minutes). Ion exchange HPLC is carded out on a Dionex Omni Pak NA1004×250 mm column at pH 12 (0.001 M NaOH) with a 2%/minute gradient of1.0M NaCI in 0.01 M NaOH. Reverse phase HPLC is carried out on aHypersil ODS 4.6×200 mm column with a minute gradient of acetonitrile in0.03M triethylammonium acetate buffer, pH 7.0. A5'-amino-oligonucleotide is prepared as described above. The 3'phosphorothioate oligonucleotide is formed as described above.

EXAMPLE 7 Synthesis of Oligonucleotide Clamps Consisting of TwoOligonucleotide Moieties in Opposite Orientations with respect to HingeRegion and/or Binding Moieties

The following oligonucleotide clamps were prepared by the proceduresdescribed above and as noted below. The 3'-bromoacetylaminooligonucleotides were prepared similarly to their 5' counterparts,except that a 3'-amino-oligonucleotide precursor was employed. The 3'phosphorothioate oligonucleotide is formed as described above. ##STR9##wherein X is --(CH₂)₆ NHC(═O)CH₂ P(═O)(O⁻⁻)S--. ##STR10## wherein X isphosphodiester. All three clamps were tested in the ATH8 assaysdescribed above.

EXAMPLE 8 Synthesis of OligonuCleotide Clamp Carrying a FluorescentLabel Attached to Hinge Region

The following oligonucleotide clamp is synthesized as described above:##STR11## wherein "pXp" indicates the presence of a branching linkage orbase, e.g. a phosphoramidate linkage, and amino derivatized cytidine, orthe like. A phosphoramidate linkage is introduced by carrying out thecoupling of one nucleoside as a hydrogen phosphonate monomer thenoxidizing the resulting phosphite linkage with I₂ and an alkyldiamine,e.g. a hexyldiamine as taught by Agrawal et al, Nucleic Acids Research,18: 5419-5423 (1990); and Jager et al, Biochemistry, 27:7237-7246(1988). This results in a free amine that can be reacted with anactivated dye.

Preferably, an AminoModifier II branching linkage (DMT--O--CH₂ CH(CH₂NH-Fmoc)-O-phosphoramidite) (commercially available from ClontechLaboratories, Palo Alto) is introduced into the hinge region. Afterdeprotection and cleavage a free primary amino group is available as anattachment site for a variety of activated fluorescent dyes, e.g. theNHS esters of fluorescein or rhodamine, commercially available fromApplied Biosystems (Foster City, CA) as TAMRA-NHS, FAM-NHS, ROX-NHS, andJOE-NHS.

EXAMPLE 9 Synthesis of Oligonucleotide Clamp Carrying a OligonucleotideAttached to Hinge Region

The oligonucleotide clamp of Example 8 is synthesized with the freeprimary amine in its hinge region. The amine is derivatized withbromoacetyl as described above. Separately, an oligonucleotide isprepared having either a 3' or 5' monophosphorothioate group, asdesired. The bromoacetylated clamp and the oligonucleotide are combinedin an aqueous solution and frozen as described above.

EXAMPLE 10 Synthesis of Oligonucleotide Clamp Dimer

The two oligonucleotide clamps shown below having free amines in theirhinge regions are separately synthesized and bromoacetylated asdescribed in Example 2. In a third synthesis, an oligonucleotide, orother linear polymeric unit, is prepared which has amonophosphorothioate group at both its 5' and 3' ends. ##STR12## wherein"pnp" represents a linkage or monomer containing an bromoacetylaminofunctionality and "p" is a phosphodiester linkage. After purification d,e, and f are combined in solution and frozen as described above. Theoligonucleotide clamp dimer is then purified by gel electrophoresis.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      AAAAGAAAAG GGGGGA16                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      CACTTTTCTTTTCC CCCCTCACACTCCCCCCTTTTCTTTTAC42                                 (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CACTTTTCTTTTGGGGGG TCACACTCCCCCCTTTTCTTTTAC42                                 (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      TTTTCTTTTCACACTTTTCTT TTGGGGGGTCACACTCCCCCC42                                 (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      TTTTTTTTTTTTCAC 15                                                            (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      ACTTTTTTTTTTTTTT 16                                                       

I claim:
 1. An oligonucleotide clamp for specifically hybridizing to atarget polynucleotide, the oligonucleotide clamp comprising:(a) at leastone oligonucleotide moiety capable of specifically hybridizing to thetarget polynucleotide with a Watson-Crick binding component and aHoogsteen- or a reverse Hoogsteen-binding component; or at least twooligonucleotide moieties designated as OL1 and OL2 linked to a hingeregion designated as G wherein at least one oligonucleotide moiety has aWatson-Crick binding component and at least one oligonucleotide moietyhas a Hoogsteen- or a reverse Hoogsteen-binding component; and (b) atleast one pair of non-oligonucleotide binding moieties, each pair ofsaid binding moieties comprising a first binding moiety and a secondbinding moiety, the first binding moiety being covalently linked to anoligonucleotide moiety and the second binding moiety being covalentlylinked to an oligonucleotide moiety, wherein a stable covalent ornon-covalent linkage is formed between the first binding moiety and thesecond binding moiety of the pair when the first and second bindingmoieties of the pair are brought into juxtaposition by the specifichybridization to the target polynucleotide of at least one or at leasttwo oligonucleotide moieties, wherein said clamp has the formula:

    X--OL1--G--OL2--Y

wherein: OL1 and OL2 are oligonucleotides specific for said targetpolynucleotide; G is a hinge region which links OL1 to OL2 so as topermit specific hybridization of OL1 and OL2 to their respective targetpolynucleotides; and X and Y are non-oligonucleotide binding moietiessuch that X and Y form a stable covalent or non-covalent linkage orcomplex whenever they are brought into juxtaposition by thehybridization of OL1 and OL2 to said target polynucleotide.
 2. Theoligonucleotide clamp of claim 1 further comprising one or more hingeregions for covalently linking two or more of said oligonucleotidemoieties.
 3. The oligonucleotide clamp of claim 1 wherein:OL1 and OL2have the same length and are from 6 to about 40 nucleotides in length:and G is defined by the formula:

    (M--L).sub.n

wherein: n is between 1 and 10; L is a linking group; and M is astraight chain, cyclic, or branched organic molecular structurecontaining from 1 to 20 carbon atoms and from 0 to 10 heteroatomsselected from the group consisting of oxygen, nitrogen, and sulfur. 4.The oligonucleotide clamp of claim 3 wherein:M is an alkyl, alkoxy,alkenyl, or aryl containing from 1 to 16 carbon atoms; it heterocyclichaving from 3 to 8 carbon atoms and from 1 to 3 heteroatoms selectedfrom the group consisting of oxygen, nitrogen, and sulfur; a glycosyl;or a nucleosidyl; and L is a phosphorus(V) linking group.
 5. Theoligonucleotide clamp of claim 4 wherein:M is an alkyl, alkoxy, alkenyl,or aryl containing from 1 to 8 carbon atoms; a glycosyl; or anucleosidyl; L is selected from the group consisting of phosphodiester,phosphotdester, methyl phosphonate, ethyl phosphonate, phosphorothioate,phosphorodithioate, and phosphoramidate; and n is from 4 to
 8. 6. Theoligonucleotide clamp of claim 5 wherein X and Y form a hydrophobiccomplex.
 7. The oligonucleotide clamp of claim 6 wherein X and Y areselected from the group consisting of alkanes, fatty acids, fattyalcohols, steroids, waxes, and fat-soluble vitamins.
 8. Theoligonucleotide clamp of claim 7 wherein X and Y are each aperhydrocyclopentanophenanthrene having from 19 to 30 carbon atoms andfrom 0 to 6 oxygen atoms.
 9. The oligonucleotide clamp of claim 8wherein X and Y are each cholesterol.
 10. The oligonucleotide clamp ofclaim 5 wherein X and Y form a covalent linkage.
 11. The oligonucleotideclamp of claim 10 wherein X is selected from the group consisting ofphosphorothioate and phosphorodithioate and wherein Y is selected fromthe group consisting of haloacyl- or haloalkylamino.
 12. Theoligonucleotide clamp of claim 10 wherein X and Y are thiophosphoryl.13. The oligonucleotide clamp of claim 1, wherein said G is 4 to 8nucleotides.
 14. The oligonucleotide clamp of claim 1, wherein said OL1is a Watson-Crick binding component and said OL2 is a Hoogsteen- or areverse-Hoogsteen binding component; or said OL1 is a Hoogsteen- or areverse-Hoogsteen binding component and said OL2 is a Watson-Crickbinding component.